The present invention provides methods and compounds useful for modifying the solubilities of oligonucleotides and their analogs. The present invention further provides methods for purifying oligonucleotides from mixtures containing the oligonucleotides and at least one contaminant wherein the contaminant is an oligonucleotide having at least one abasic site.
Modern therapeutic efforts are generally focused on the functions of proteins which contribute to many diseases in animals and man. There have been numerous attempts to modulate the production of such proteins by interfering with the function of biomolecules, such as intracellular RNA, that are involved in the synthesis of these proteins. It is anticipated that protein production will thus be inhibited or abolished, resulting in a beneficial therapeutic effect. The general object of such therapeutic approaches is to interfere with or modulate gene expression events that lead to the formation of undesired proteins.
One such method for the inhibition of specific gene expression is the use of oligonucleotides and oligonucleotide analogs as antisense drugs. These oligonucleotide or oligonucleotide analogs are designed to be complementary to a specific target messenger RNA (mRNA) or DNA, that encodes for the undesired protein. The oligonucleotide or oligonucleotide analog is expected to hybridize with good affinity and selectivity to its target nucleic acid, such that the normal essential functions of the target nucleic acid are disrupted. Antisense therapeutics hold great promise as evidenced by the large number of oligonucleotides and oligonucleotide analogs that have been evaluated clinically in recent times. Further, oligonucleotides and oligonucleotide analogs have shown significant promise in the diagnosis of disease and have also been used extensively as probes in diagnostic kits and as research reagents.
There is, therefore, a great need for the large scale production of oligonucleotides and oligonucleotide analogs for commercial application. The predominant synthetic regime currently in use for oligonucleotide synthesis is the phosphoramidite method as developed by Caruthers (Caruthers, M. H. Gene Synthesis Machines: DNA Chemistry and Its Uses. Science, 1985, 230, 281-85). The phosphoramidite method transformed oligonucleotide synthesis from a manual or semi-manual procedure carried out by a few specialists into a commercialized process performed by a machine. The oligonucleotides are synthesized on a solid-support via sequential reactions in a predetermined order, typically controlled by a computerized pumping system. The crux of this chemistry is a highly efficient coupling reaction ( greater than 98%) between a 5xe2x80x2-hydroxyl group of a support-bound deoxynucleoside and an alkyl 5xe2x80x2-O-DMTr-3xe2x80x2-O-(N,N-diisopropylamino-O-cyanoethyl)phosphoramidte deoxynucleoside. For example, oligonucleotide synthesis typically begins with a nucleoside linked to a solid-support, typically via a linker molecule attached to the 3xe2x80x2-oxygen of the first nucleosidic synthon. Deprotection (or xe2x80x9ccleavagexe2x80x9d) of the 5xe2x80x2-hydroxyl group is effected by treatment with an acid (3% dichloroacetic acid (DCA) in dichloromethane or toluene) which removes the 5xe2x80x2-O-(4,4xe2x80x2-dimethoxytriphenylmethyl) hydroxyl protecting group (DMTr) to provide an oligonucleotide having a free 5xe2x80x2-OH group. Such protecting groups are routinely used in oligonucleotide synthesis to allow selective reaction between two functional groups while protecting all other functionalities present in the reacting molecules.
The next step consists of premixing a nucleoside phosphoramidite with an activator such as 1-H tetrazole. The very reactive P(III) tetrazolide intermediate reacts almost immediately with the 5xe2x80x2-OH group of the support bound nucleoside to generate a dinucleoside phosphite with a phosphite triester internucleosidic linkage. The unstable P(III) species is oxidized to a more stable P(V) internucleosidic linkage with iodine to the phosphotriester before proceeding with chain extension. A capping reaction with an acylating reagent is performed to prevent the unreacted 5xe2x80x2-OH groups from further extension. These steps are then repeated iteratively until the desired oligonucleotide is obtained. A more detailed treatment of oligonucleotide synthesis, and further representative synthetic procedures can be found in Oligonucleotides And Analogues A Practical Approach, Eckstein, F., Ed., IRL Press, N.Y, 1991.
One challenge facing commercialization of oligonucleotide based therapeutic and diagnostic products is the ability to manufacture and market these products at a reasonable cost and with a high level of oligonucleotide purity.
The trityl group has been used for the temporary protection of primary hydroxyl groups due to the generally good crystallizing properties imparted by the trityl ether and its easy removal through mild acid treatment or by hydrogenolysis (Agarawal, K. I.; Yamazaki, A.; Cashion, P. L.; Khorana, H. G., Angew Chem. Int Ed. Engl. 1972, 451 and Stanek, J. Top. Curr. Chem. 1990, 54, 234). However, the literature indicates that detritylation is a problematic operation. Low yields, formation of by-products, acyl migration and glycosidic bond cleavage or depurination often arise from protic acid-catalyzed detritylation reactions (e.g., 80% acetic acid acid at reflux (Micheel, F., Ber. 1932, 65, 262), 80% formic acid in ethyl acetate at room temperature (Soudheimer, S. J.; Eby, R.; Schuerch, C., Carbohydr. Res. 1978, 60, 187 and Bessodes, M.; Komiotis, D.; Antonakis, K., Tetrahedron Lett. 1986, 27, 579), hydrogen chloride in methanol (Verkade, P. E.; Vander Lee, J.; Meerburg, W. Rec. Trav. Chim., 1935, 54, 716) or other solvent (Choy, Y. M.; Unrau, A. M., Carbohydr. Res. 1971, 17, 439), and hydrogen bromide in acetic acid (Roy, N.; Timell, T. E., Carbohydr. Res. 1968, 7, 82 and Barker, G. R., Methods Carbohydr. Chem. 1963, 2, 68), among others (Helferich, B., Adv. Carbohydr. Chem. 1948, 3, 79).
The deprotection of a trityl group is usually performed under acidic conditions using a protic or a Lewis acid in an organic solvent. Alternate deprotection protocols have been attempted such that acidic conditions are avoided in an attempt to ameliorate the problems of depurination in purine rich oligonucleotides. These techniques, however, have not been reported to have been successfully applied to the large scale manufacture of oligonucleotides, as is required for research or commercial purposes.
The importance of a good separation technique for synthetic oligonucleotides is often neglected. The impurities from a large number of reactions are stored upon the support and must all be resolved, preferably in a single step. Powerful separation methods have been developed to purify oligonucleotides. For example, polyacrylamide gel electrophoresis separates oligonucleotides by virtue of their charge differences. Other techniques include high performance liquid chromatography (HPLC), including ion exchange chromatography which resolves by charge differences and reverse phase chromatography which separates according to hydrophobicty. These chromatographic techniques, however, have their limitations. These limitations are either that the purification is limited to milligram quantities of materials, or there is an insufficient resolution between the desired oligonucleotide and the contaminant impurity.
For the foregoing reasons, there exists a need for new methods that address the shortcomings of the large scale production and purification of oligonucleotides as discussed above.
The present invention is directed to methods and compounds useful for purifying oligonucleotides and oligonucleotide analogs from mixtures having contaminants comprising oligonucleotides or oligonucleotide analogs that have undesired abasic sites. Mixtures are treated with amino reagents reactive with and capable of forming imine linkages with the contaminants. Chemical modification of undesired contaminants using the present method enhances separation of the desired oligonucleotides from undesired contaminants by methods that are ineffective for the parent mixtures prior to such modification.
In preferred embodiments, the imine-linked contaminants are separated from the oligonucleotide to be purified using chromatography.
In other preferred embodiments, the imine-linked contaminants are separated from the oligonucleotide to be purified by selectively precipitating the imine-linked contaminants with respect to the oligonucleotide to be purified.
In other preferred embodiments, the imine-linked contaminants are separated from the oligonucleotide to be purified by selectively precipitating the oligonucleotide to be purified with respect to the imine-linked contaminant.
In other preferred embodiments, the imine-linked oligonucleotides are separated from the oligonucleotide to be purified by a liquid-liquid extraction.
In some preferred embodiments, the imine-linked contaminants are separated from the oligonucleotide to be purified using chromatography with a single solvent, or with two or more miscible solvents.
In other preferred embodiments of the invention, the imine-linked contaminants oligonucleotides are separated from the oligonucleotide to be purified by precipitation using two or more immiscible solvents, or two or more miscible solvents.
In other preferred embodiments, the imine-linked contaminants are separated from the oligonucleotide to be purified by liquid-liquid extraction using two or more immiscible solvents.
In a preferred embodiment of the invention, the imine-linked contaminants are separated from the oligonucleotide to be purified based upon differences in solubility of the oligonucleotide and the imine-linked contaminant in a selected solvent.
In one preferred embodiment, the imine-linked contaminants are more soluble in a selected solvent than the oligonucleotide to be purified. In another preferred embodiments, the imine-linked contaminants are less soluble in a selected solvent than the oligonucleotide to be purified.
In one preferred embodiment of the invention, the difference in solubility is a difference wherein the oligonucleotide to be purified is more soluble in a first solvent, preferably water or an aqueous solvent, than the imine-linked contaminants and the imine-linked contaminants are more soluble in a second solvent, preferably an organic solvent, than the oligonucleotide to be purified and the first and second solvents are immiscible. In further preferred embodiments, the organic solvent includes benzene, diethyl ether, ethyl acetate, hexane, pentane, chloroform, dichloromethane, carbon tetrachloride, and the like. In other preferred embodiments the first and second solvents are miscible and the second solvent is preferably an organic solvent that is miscible with water such as, acetone, methanol, isopropanol, ethanol and the like.
In preferred embodiments, the amino reagents include amines, hydrazines, hydroxylamines, semicarbazides, and thiosemicarbazides.
In one preferred embodiment of the present invention, the amino reagent is linked to a polymeric support thereby forming a linked amino reagent. The amino reagents may be linked to solid-phase polymeric supports, to form, for example, a hydroxylamine resin, or may be linked to liquid-phase support, preferably hydrophilic supports. In preferred embodiments, the liquid-phase polymeric support is a polyvinyl alcohol, a polyethylene glycol (PEG), a cellulose, or a polyvinyl alcohol-poly(1-vinyl-2-pyrrolidinone). Preferred liquid-support linked amino reagents are polyethylene glycol (PEG) amine, polyethylene glycol (PEG) hydrazine, polyethylene glycol hydroxylamine, polyethylene glycol semicarbazide, and polyetheylene glycol thiosemicarbazide.
In further embodiments the amino reagent may include a surfactant, such as a non-ionic surfactant. A preferred amino reagent including such a surfactant has the formula: 
wherein:
x is from 0 to 20,
y is from 0 to 5,
n is from 0 to 150; and
Z is xe2x80x94NH2, xe2x80x94NHNH2, xe2x80x94ONH2, xe2x80x94NHC(O)NHNH2 or xe2x80x94NHC(S)NHNH2.
In a preferred embodiment x is 8, y is 1 and n is 12.
In some preferred embodiments of the present invention, methods for purifying an oligonucleotide from a mixture wherein the mixture includes the oligonucleotide and at least one contaminant, wherein the contaminant comprises at least one aldehyde moiety, comprise the steps of:
treating the mixture with a compound of formula I: 
xe2x80x83wherein:
x is from 0 to about 20, preferably 8;
y is from 0 to about 5, preferably 1;
n is from 0 to about 150, preferably 12; and
Z is a reactive nitrogenous moiety, such as xe2x80x94NH2, xe2x80x94NHNH2, xe2x80x94ONH2, xe2x80x94NHC(O)NHNH2, or xe2x80x94NHC(S)NH2, that is capable of reacting with an aldehyde to form an imine;
for a time and under conditions effective to form imine linkages with each contaminant; and
separating said oligonucleotide from said imine linked contaminants.
In some preferred embodiments of the present invention, methods for purifying an oligonucleotide from a mixture wherein the mixture includes the oligonucleotide and at least one contaminant, wherein the contaminant comprises at least one aldehyde moiety, comprise the steps of:
treating the mixture with a plurality of compounds of formula I: 
xe2x80x83wherein the compounds differ with respect to the value of n, and wherein all other variables are as described above.
In another aspect of the invention, a method is provided for modifying the solubility of an oligonucleotide comprising:
selecting an oligonucleotide having at least one abasic site;
treating the oligonucleotide with an amino reagent reactive with the oligonucleotide for a time and under conditions effective to form an imine linkage between the abasic site of the oligonucleotide and the amino reagent.